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Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan Alaa M. Ibrahim, Ibrahim A. Adam, Badreldin T. Osman, Imadeldin E. Aradaib ∗ Molecular Biology Laboratory (MBL), Department of Clinical Medicine, Faculty of Veterinary Medicine, University Khartoum, Sudan
a r t i c l e
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Article history: Received 7 August 2014 Received in revised form 24 November 2014 Accepted 4 March 2015 Available online xxx Keywords: Epidemiology Cattle Crimean-Congo hemorrhagic fevers ELISA Sudan
a b s t r a c t Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by CCHF virus (CCHFV) of the genus Nairovirus in the family Bunyaviridae. CCHFV causes subclinical infection in domestic livestock and an often fatal hemorrhagic illness in humans, with approximately 30% mortality rates. In the present study, a cross-sectional serosurvey was conducted in a total of 282 randomly selected cattle from five localities in East Darfur State, Sudan. The exposure status to CCHF was determined using enzyme-linked immunosorbent assay (ELISA) for detection of CCHFV-specific IgG antibodies in cattle serum samples. The CCHFV-specific IgG antibodies were detected in 54 out of 282 animals, accounting for a 19.14% prevalence rate. Older cattle (>2 years of age) were approximately five times more likely to be infected with the virus (OR = 4.90, CI = 1.28–18.98, p-value = 0.02). Heavily tick-infested cattle (ticks all over the body) were at 11 times higher at risk compared to tick-free animals (OR = 11.11, CI = 2.86–43.25, p-value = 0.01). Grazing system is another factor affecting CCHF, where cattle grazing on open system were 27 times more at risk compared to other grazing systems (OR = 27.22, CI = 7.46–99.24, p-value = 0.001). There was an association between localities and CCHF cattle (OR = 0.24, CI = 0.07–0.83, p-value = 0.02). This study confirms the exposure of cattle to CCHF in East Darfur and identifies potential risk factors associated with the disease. Further epidemiological studies and improved surveillance are urgently needed to prevent a possible outbreak of CCHF among humans in the Darfur region of Sudan. © 2015 Elsevier GmbH. All rights reserved.
Introduction Crimean-Congo hemorrhagic fever (CCHF), caused by CCHF virus (CCHFV), is a zoonotic tick-borne disease, which is transmitted to humans by tick bites, handling of ticks, exposure to blood or tissues of viremic livestock, or direct contact with blood and bodily fluids of infected patients (Swanepoel et al., 1987; Vesenjak-Hirjan et al., 1991; Schwarz et al., 1995, 1996; Rodriguez et al., 1997). CCHF outbreaks constitute a threat to public health because of its epidemic potential, the severity of disease it causes, its potential to cause nosocomial outbreaks, and the limited options available for treatment and management of infected patients (Altaf et al., 1998; Burt et al., 1998; Ergonul, 2006; Whitehouse, 2004; Avsic-Zupanc, 2007). Recently, CCHFV has been repeatedly reported as an important emerging infectious viral pathogen in the Kordufan region, Sudan. In Sudan, the first outbreak of CCHF was reported in 2008 among health care workers in Alfulah rural hospital, West Kordufan (Aradaib et al., 2010). Subsequently, another outbreak was
∗ Corresponding author. Tel.: +249 912380932; fax: +249 185312638. E-mail address: [email protected] (I.E. Aradaib).
reported in 2009 in Donkup village, Abyei District, South Kordufan (Aradaib et al., 2011). In February, 2010, a nosocomially acquired CCHFV infection was reported in an attending physician in North Kordufan as a result of providing medical care to CCHF infected patient from Lagawa District, an area of endemicity in the State of South Kordufan. However, CCHF has never been recognized in the Darfur region of the Sudan (Elata et al., 2011). Serological studies have suggested the presence of various arboviruses in Sudan, including CCHFV (Watts et al., 1994; McCarthy et al., 1996). Indirect serological evidence of CCHF infection was recorded in camels exported from Sudan to Egypt (Morrill et al., 1990) and in sheep and goats exported to Saudi Arabia (Hassanein et al., 1997). Recently, we reported an overall prevalence rate of 7% among cattle in the State of North Kordufan (Adam et al., 2013). It is well documented that viremia and CCHFV-specific antibodies develop in infected livestock including, sheep, cattle and camels as well as in domestic donkeys (Swanepoel et al., 1987; Lwande et al., 2012). However, the infection is subclinical and no clinical hemorrhagic disease appears to be associated with CCHFV in infected animals (Burt et al., 1993). Nevertheless, cattle can amplify the virus and will become seroconverted. However, they do not show any signs of clinical hemorrhagic disease indicating
http://dx.doi.org/10.1016/j.ttbdis.2015.03.002 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Ibrahim, A.M., et al., Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.002
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subclinical infection. Therefore, at the time of viremia infected cattle could provide a potential source of infection to infested ticks. In addition, viremic cattle could also provide infection to humans, particularly after slaughtering and subsequent contact with their infected tissues including blood or meat. Therefore, it is suggested that these animals play an important role in the epidemiology of the disease (Shepherd et al., 1987; Garcia et al., 2006; Martin et al., 1987). Currently, it is becoming increasingly obvious that the control of emerging viral pathogens, such as CCHFV, is especially important in the Sudan given the large numbers of livestock in the country, and their importance to the national economy and rural communities. To better predict and respond to CCHF in Darfur region, further epidemiologic studies including implementation of improved surveillance, for such an emerging viral pathogen, are urgently needed (Martin et al., 1987). The present study was conducted to estimate the prevalence and to identify the potential risk factors associated with CCHF among cattle in East Darfur State, Sudan. Materials and methods Study area The State of East Darfur is located between latitude 12–9.5 N–longitude 25–28 E and altitude of 449 m (1476 feet) above sea level and about 831 km from Khartoum, the capital of Sudan. The State is boarded by North Darfur State in the north; Northern Kordufan in the east, South Darfur in the west, and is bordered by the State of South Sudan to the south. Total population in East Darfur State is approximately 0.3 million as estimated in 2006. The livestock population in East Darfur constitutes one of the major sources of the income to rural communities and the national economy, at large. East Darfur State is one of the main animal rearing areas in the country and possesses large numbers of live stock. The cattle population in this state is presently estimated to be as 2–3 million, 80% of which are kept under nomadic system, where nomads migrate continuously to south in the dry season and back to north in the rainy season. During this movement the nomads cover areas at the borders of the State of South Sudan up to north Darfur, where the animals inter mix freely and share common water sources, pasture route and premises with cattle in the State of South Sudan. The map of the State of East Darfur is shown in Fig. 1. Study design A cross sectional study was conducted to estimate the prevalence rate of CCHFV group-specific IgG antibodies in cattle and to investigate potential risk factors associated with the disease. The multistage probability sampling method was conducted. Five localities were randomly selected from the State of East Darfur. Two administration units were selected from each locality. Seven villages were selected from each unit. Finally, simple random sampling was applied to choose the animals from each herd. Questionnaire A pre-tested structured questionnaire with the primary objective of elucidating the multifactorial background of disease was conducted in an interactive manner at all selected herds. All animals included in this study were subjected to a questionnaire, which was filled out by the animal owners. The questionnaire included individual risk factor attributes including age (younger animals <2 years, older animals 2 years and above), sex (male, female), breed (indigenous, cross), body condition (thin, emaciated, fat), and management risk factor attributes including herd size (small, medium,
Fig. 1. Map of the State of East Darfur, Sudan.
large), grazing system (nomadic, semi-nomadic, stationary), tick vector (presence or absence), the source of each animal in the herd (raised on farm, purchased from other farms or purchased from local market) and the five localities included in the study. Ethical clearance The blood collection procedure from cattle was performed by qualified veterinarians following proper physical restraint of animals to ensure both personnel and animal safety. Livestock owners were explained the study purposes and procedures and upon agreeing to participate, they provided a written consent prior to study procedures and blood collection from their animals. The study received ethical clearance from the Research Board of the College of Veterinary Medicine, Sudan University for Science and Technology, Khartoum, Sudan. The risk factor information was obtained from the animal owners through the questionnaire form, which permitted use of the blood samples for diagnostic and research purposes. Collection of blood samples A total of 282 blood samples of cattle were collected randomly from five localities in East Darfur State, Sudan. These localities include (ElDdeain, AbuJabra, Assalaya, Elfirdus, and Bahr Alarab). Blood samples were collected by qualified veterinarians from the jugular veins in clean sterile vaccutainers and were allowed to clot and sera were separated and kept frozen at −20 ◦ C until used. Enzyme-linked immunosorbent assay (ELISA) Indirect enzyme-linked immunosorbent assay (ELISA) was performed to screen the sera for CCHFV-specific IgG antibodies basically as described (Adam et al., 2013). ELISA was performed in 96-well immunoassay microplates (Nunc, Roskilde, Denmark) and optimal working dilutions of reagents were determined by
Please cite this article in press as: Ibrahim, A.M., et al., Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.002
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chessboard titration. Unless stated otherwise, 100 uL test volumes were used. Incubations were performed for 1 h at 37 ◦ C. The plates were washed three times with PBS containing 1.0% Tween 20 (Merck, Darmstadt, Germany) (PBST), wells were post-coated with 200 l of PBS containing 2% bovine serum albumin (Calbiochem, La Jolla, USA), and the diluents for the reagents was PBS containing 10% Skimmed milk (Amba, Denmark). Briefly, the plates were coated with sucrose-acetone extracted CCHFV antigen and incubated overnight at 4 ◦ C. The source of the antigen used is cell lysate from CCHFV Nigeria strain IbAr 10200. The antigen used in this study was obtained from the Center for Disease Control and Prevention, Atlanta, USA). The plates were washed, and aliquots of test sera (positive and negative controls) were added in separate wells at a dilution of 1:40. After a 1-h incubation, the plates were washed, and rabbit anti-bovine IgG conjugated with horse radish peroxidase (HRP) was added to the plate at a dilution of 1: 2000 and incubated for 1 h. The plates were then washed and the substrate, 2,22-azino-bis (3-ethylbenthiazoline-6-sulfonic acid (Kirkegaard and Perry Laboratories) was added. A positive CCHFV-infected cattle serum sample was incorporated in each ELISA plate as positive control to estimate the higher limit of the sensitivity. Negative control sera were obtained from CCHF-free animals and from cattle infected with Rift Valley fever virus (RVFV), a related viral hemorrhagic fever virus, to estimate the lower level of the specificity of the ELISA assay. The results were read using ELISA reader set at 405 nm. A presumptive diagnosis was made when IgG antibodies in the test sample had a significant color change or had higher optical density than the ratio between the positive and negative controls. Statistical analyses The data collected were entered into a computer on a Microsoft Excel spreadsheet. Statistical analysis was performed using ‘Statistical package for the social sciences’ (SPSS), version 16 (for Windows). Associations between the outcome variable (status of CCHFV-specific antibodies in cattle) and its potential risk factors were first screened in a univariable analysis using Chi-square test. Potential risk factors with p-value <0.25 (two tailed; ˛ = 0.25) were initially considered significant in 2 test. The results of the univariable analysis were reentered in the final model using multivariable analysis. A multivariable model for the outcome variable was constructed using manual stepwise forward logistic regression analysis. CCHF was considered as the dependent variable and the risk factors as independent variables. Finally, odd ratios and 95% confidence interval (CI) were calculated, and risk factors with a p-value <0.05 were taken as significant association to CCHFV infection. Results The result of this survey showed that 54 out of 282 cattle serum samples were found to be positive for CCHFV-specific-IgG antibodies, accounting for an overall prevalence rate of a 19.14% among cattle in East Darfur State, Sudan. Univariate analysis using Chi-square test was conducted for the association between the potential risk factors and CCHFV infection and p-value (p ≤ 0.25) was initially considered significant. The results of the univariate analysis showed that the independent variables including, age, animal source, tick number, body condition, grazing system, herd size, tick treatment frequency and locality were statistically significant (Table 1). The risk factors that were significant in the univariable model were re-entered into a final multivariate model using logistic regression analysis. In the final models, a variable with a p-value <0.05 was considered statistically significant. The individual risk factors attributes indicated that the older cattle (>2 years of age)
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were approximately five times more likely to be infected with the virus (OR = 4.90, CI = 1.28–18.98, p-value = 0.02). There was an association between locality (Barelarab) and CCHFV infected cattle (OR = 0.24, CI = 0.07–0.83, p-value = 0.02). The management risk factors attributes showed that heavily tick-infested cattle (ticks all over the body) were eleven time higher at risk compared to tick-free animals (OR = 11.11, CI = 2.86–43.25, p-value = 0.01). In addition, the type of the grazing system has significant association with CCHFV seropositivity. Cattle grazing on an open system were 27 times more likely to be at risk for CCHF compared to those grazing on a close system (OR = 27.22, CI = 7.46–99.24, p-value = 0.001). The results are summarized in (Table 2). In contrast, there was no significant association between CCHFV seropositive cattle and other individual or management risk factors included in the study such as animal sex, breed, body condition, animal source, herd size and tick treatment.
Discussion Crimean-Congo hemorrhagic fever is a tick-borne viral disease mainly affecting nomads who live in close contact with tickinfested livestock, namely Hyalomma ticks, the principal vectors of the virus. CCHFV causes an often fatal hemorrhagic illness in humans and asymptomatic infection in domestic livestock. In rural communities, tick-infested donkeys were considered to be potential source of CCHF in humans (Lwande et al., 2012). Therefore, it is suggested that livestock and domestic donkeys play an important role in the epidemiology of the disease. The occurrence of the periodic outbreaks and sporadic cases of CCHF in endemic countries necessitate the importance of improved surveillance system for this important emerging viral pathogen (Altaf et al., 1998; Aradaib et al., 2010, 2011; Burney et al., 1980; Ahmeti and Raka, 2006; Gurbuz et al., 2009; Khan et al., 1997). In this study, cattle were selected as CCHFV infection appears to occur most frequently in larger mammals, which are the preferred hosts of adult tick vector, Hylomma species (Shepherd et al., 1998). We believe the occurrence of multiple CCHF outbreaks in region in the past few years, and the risks these cases pose for medical staff in resource poor health care facilities indicate the importance of improved surveillance in Sudan for this important disease. The Darfur region of Sudan was thought to be CCHF-free zone and infection with CCHFV has never been reported in the region. This study expands on existing data indicating that CCHF, known to be endemic in Africa, is broadly distributed within Sudan. In addition, our investigation illustrates that CCHF has been reported for the first time among cattle in the Darfur region, Sudan. More over, collection of bovine blood samples for generation of the present data was made available despite the violence in the region. It is worth mentioning that Darfur region shares boarders with four different countries including Chad, Libya, Central African Republic and State of South Sudan and it would be interesting to trace the movement of the virus in the region. Further more, the results of this epidemiological study will stimulate further molecular characterization studies such as viral genome sequencing and subsequent phylogeny to determine the virus genetic lineages circulating in the region. Our result indicated that, the overall prevalence of CCHFV IgG-specific antibodies was 19.14%. The CCHFV IgG-specific antibodies recorded in this study showed evidence of prior exposure to CCHF by cattle population in East Darfur State, Sudan. The high prevalence rate (19.14%) indicates significant circulation of CCHFV among cattle of East Darfur and that human population in the region may have also been infected with CCHFV. However, acute CCHF cases have never been diagnosed among humans in Darfur region of the Sudan. This is probably attributed to the fact that
Please cite this article in press as: Ibrahim, A.M., et al., Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.002
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Table 1 Univariate analysis for the association between potential risk factors and CCHFV infection among cattle in East Darfur State of Sudan using Chi-square test. Risk factors
Animals tested
Locality Aldeain Abu Jabra Bahr Alarab Assalaya Elfirdus
Animals affected (%)
74 49 99 23 37
16 (21.6%) 9 (18.4%) 13 (13.1%) 4 (17.4%) 12 (32.4%)
Age Small Old
67 215
3 (4.5%) 51 (23.7%)
Sex female male
220 62
42 (19.1%) 12 (19.4%)
Breed Endigenous Cross
265 17
52 (19.6%) 2 (11.8%)
Body condition Fat Thin
20 262
2 (10%) 52 (19.8%)
Animal source -Raised on farm Purchased from other farm Purchased from local market
206 50 26
35 (17%) 12 (24%) 7 (26.9%)
Grazing system Stationary Nomadic
112 170
3 (2.7%) 51 (30%)
Herd size Small Medium Large
48 119 115
5 (10.4%) 25 (21%) 24 (20.4%)
Tick treatment frequency 1–3 months More than 3 months
80 202
18 (22.5%) 36 (17.8%)
Tick number No Small Large
75 112 85
5 (6.7%) 24 (19.7%) 25 (29.4%)
Table 2 Multivariate analysis using logistic regression model for significant association (p < 0.05) between risk factors and CCHFV infection among cattle in East Darfur State, Sudan. Risk factors
OR
95% CI
p-value
Age
Small Old
Ref 4.90
1.28–18.98
0.02
Localities
Aldeain Abugabra Bahrelarab Asalaya Fardus
Ref 0.65 0.24 0.75 1.51
0.18–2.44 0.07–0.83 015–3.89 0.42–5.50
0.52 0.02 0.73 0.53
Stationary Nomadic
Ref 27.22
7.46–99.24
0.01
No Small Large
Ref 6.05 11.11
1.72–21.33 2.86–43.25
0.01 0.01
Grazing system Tick number
several acute febrile illnesses such as malaria, brucellosis, typhoid and other related hemorrhagic fevers are endemic in the region. Under these circumstances, it would be extremely difficult, if not impossible, for health care workers to differentiate CCHF from other endemic febrile illness based on clinical presentation. Recently, a lot of research efforts have been conducted in our laboratory to facilitate rapid detection and differentiation of arbovirus infections with special emphasis on CCHF during outbreaks of the disease in the Kordufan region of Sudan (Aradaib et al., 1998, 2005, 2010, 2011,
df
2
p-value
4
6.89
0.14
1
12.23
0.01
1
0.01
0.55
1
0.64
0.33
1
1.16
0.22
2
2.40
0.13
1
32.55
0.01
2
2.85
0.24
1
0.81
0.23
2
13.35
0.01
2013). Previous CCHF studies in humans showed prevalence rate of 13.1% among nomadic tribes in Senegal (Chapman et al., 1991). In addition, a serosurvey on CCHF among pastoralists in Ijara District, east Northern province, Kenya, showed seroprevalence rates of 23 and 14% in Sangailu and Ijara regions, respectively (Lwande et al., 2012). Using the multivariate logistic regression analysis, only four independent risk factors were associated with CCHFV seropositivity in cattle and were considered statistically significant p-value <0.05. There was significant difference between CCHFV infection rate and the age of the animal. When assessing age as risk factor, it was shown that the calves started to get infected with CCHFV after the age of 2 years. At this age, the animals are usually released into the pasture for grazing, where they are likely to be exposed to infected tick and subsequent CCHFV infection. We believe that the association of CCHFV infection rate and age is probably attributed to frequent exposure of older cattle to infected tick in the pasture. In contrast, young calves are usually kept indoors and are well taken care of by the animal owners from infectious diseases particularly, tick-borne infections. It should be noted that the CCHFV-specific antibodies detected among cattle in East Darfur state indicate natural infection as there is no vaccination program for this disease. In addition, all cattle included in this study aged over one year. Therefore, it is assumed that maternal antibodies no longer persisted and that antibody indicated direct exposure to CCHFV. Previous epidemiological surveys indicated that there were higher risks of older cattle for CCHF in different countries including Sudan, Egypt, Iran and Turkey (Adam et al., 2013; Garcia et al., 2006; Mohamed
Please cite this article in press as: Ibrahim, A.M., et al., Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.002
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et al., 2008; Lotfollahzadeh et al., 2011). In addition, the study revealed significant association between heavily tick-infested cattle and CCHFV seropositivity. It is well documented that CCHF is a tick-borne zoonotic disease and that heavily tick-infested cattle are likely to become CCHFV positive by bites of infected ticks (Adam et al., 2013; Mohamed et al., 2008; Swanepoel et al., 1985). Moreover, there was significant association between the open system of grazing and CCHFV seropositive cattle. Frequent exposure of cattle to the pasture results in contact with many tick-infested species of mammals, which can transmit the virus (Swanepoel et al., 1983). It should also be noted that treatment of cattle with insecticides should be applied monthly to prevent tick infestation. Small vertebrates such as hares and hedgehogs, which are infested by immature ticks, may be particularly important as amplifying hosts (Swanepoel et al., 1987). The ground-based birds such as ostrich can amplify the virus; others are refractory to infection but may act as mechanical vectors by transporting infected ticks over a long distance. Migratory birds might spread the virus between distant geographic areas (Deyde et al., 2006; Lindeborg et al., 2011). Furthermore, there was a significant difference between localities and CCHFV seropositivity. The highest and lowest rates of CCHFV seropositivity were recorded in Bahr Elarab and Asalaya, respectively. The high level of CCHFV infection in Bahr Elarab is attributed to the dry climate and high variations in temperatures in the region. In contrast, the risk assessment studies indicated that there was no significant association between CCHF and the rest of the individual or management risk factors included in the study. It is worth mentioning that the gender has no significant association with CCHFV seropositivity as both sexes are equally infected with the disease. The lack of association between CCHF and tick treatment is probably due to the infrequent treatments with insecticides by the nomads specially, under field conditions. Conclusions This study confirms the transmission of CCHFV among cattle in East Darfur State. Age, locality, open system of grazing, and tick infestation are potential risk factors for CCHF. Human populations in East Darfur State are at risk of contracting the infection with the virus. The residents should also be educated about the risks of the disease, and prevention of the infection through tick control. In this regard, effective animal husbandry and management system should be applied to control tick infestations in domestic livestock populations. Health care providers should be aware of the disease and be prepared for a possible outbreak in the region. Physicians and medical health workers in East Darfur State should consider this virus in their efforts to diagnose the disease in patients with clinical presentations compatible with those of CCHF. Future surveillance program for CCHF should be extended to include other susceptible animals such as sheep, goats, camels and donkeys. In addition, the high-risk human groups (Veterinarians, animal attendants, slaughter house workers and butchers), and the distribution of ticks in the region should also be considered to better predict and respond to CCHF outbreak in the State of East Darfur, Sudan. Author details Molecular Biology Laboratory (MBL), Department of Clinical Medicine, Faculty of Veterinary Medicine, University of Khartoum, P.O. Box 32 Khartoum North, Sudan. Authors’ contributions AMI designed the study, optimized the ELISA for detection of IgG antibodies in cattle sera, performed the statistical analysis, and
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prepared the draft manuscript. IAA collected blood samples and designed the study. IEA designed the experiment and prepared the final manuscript. All authors read and approved the final version of the manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgements The authors would like to thank Dr. Stuart T. Nichol of the Molecular Biology Laboratory, Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, USA, for provision of CCHFV antigen. The technical assistance of Dr. Shakir Z. Bushara; Dr. Mubarak Mahmoud and Mr. Abdalla M. Fadlelmoula is gratefully acknowledged. This study received partial financial support from the University of Khartoum, Sudan. The result of this study does not reflect the opinion of the funding sources. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.03.002 References Adam, I.A., Mahmoud, M.A.A., Aradaib, I.E., 2013. A seroepidemiological survey of Crimean Congo hemorrhagic fever among Cattle in North Kordufan State, Sudan. Virol. J. (10), 178, http://dx.doi.org/10.1186/1743-422X-10-178 Ahmeti, S., Raka, L., 2006. Crimean-Congo haemorrhagic fever in Kosova: a fatal case report. Virol. J. 3, 85. Altaf, A., Luby, S., Ahmed, A.J., Zaidi, N., Khan, A.J., Mirza, S., Mc Cormick, J., FisherHoch, S., 1998. Outbreak of Crimean-Congo haemorrhagic fever in Quetta, Pakistan: contact tracing and risk assessment. Trop. Med. Int. Health 3, 878–882. Aradaib, I.E., Erickson, B.R., Karsany, M.E., Khristova, M.L., Elageb, R.M., Mohamed, M.E.H., Nichol, S.T., 2011. Multiple Crimean-Congo hemorrhagic fever virus strains are associated with disease outbreaks in Sudan, 2008–2009. PLoS Negl. Trop. Dis. 5, e1159, http://dx.doi.org/10.1371/journal.pntd.0001159 Aradaib, I.E., Erickson, B.R., Mustafa, M.E., Khristova, M.L., Saeed, N.S., Elageb, R.M., Nichol, S.T., 2010. Nosocomial outbreak of Crimean-Congo hemorrhagic fever, Sudan. Emerg. Infect. Dis. 16, 837–839. Aradaib, I.E., Mohamed, M.E.H., Abdalla, T.M., Abdalla, M.Y., Abdalla, M.A., Sarr, J.A., Karrar, A.E., 2005. Serogrouping of United states and some African serotypes of Bluetongue virus using RT-PCR. Vet. Microbiol. 111, 125–128. Aradaib, I.E., Mohammed, M.E.H., Ahmed, S., Ibrahim, K.E.E., Yilma, T.D., Karrar, A.E., Cullor, J.S., Osburn, B.I., 1998. A multiplex PCR for simultaneous detection and identification of United States serotypes of Epizootic haemorrhagic disease virus. Sud. J. Vet. Sci. Anim. Husb. 37, 1–12. Aradaib, I.E., Erickson, B.R., Karsany, M.S., Khristova, M.L., Elageb, R.M., Mohamed, M.E., Nichol, S.T., 2013. Rift Valley fever, Sudan, 2007–2010. Emerg. Infect. Dis. 19, 45–52. Avsic-Zupanc, T., 2007. Epidemiology of Crimean-Congo hemorrhagic fever in the Balkans. In: Ergonul, O.W.C.A. (Ed.), Crimean-Congo Hemorrhagic Fever: A Global Perspective. Springer, Dordrecht, pp. 75–88. Burney, M.I., Ghafoor, A., Saleen, M., Webb, P.A., Casals, J., 1980. Nosocomial outbreak of viral hemorrhagic fever caused by Crimean hemorrhagic fever-Congo virus in Pakistan in 1976. Am. J. Trop. Med. Hyg. 29, 941–947. Burt, F.J., Leman, P.A., Smith, J.F., Swanepoel, R., 1998. The use of a reverse transcription-polymerase chain reaction for the detection of viral nucleic acid in the diagnosis of Crimean-Congo haemorrhagic fever. J. Virol. Methods 70, 129–137. Burt, F.J., Swanepool, R., Braack, L.E.G., 1993. Enzyme-linked immunosorbent assays for the detection of antibody to Crimean-Congo haemorrhagic fever virus in the sera of livestock and wild vertebrates. Epidemiol. Infect. 111, 547–557. Chapman, L.E., Wilson, M.L., Hall, D.B., 1991. Risk factors for Crimean-Congo hemorrhagic fever in rural northern Senegal. J. Infect. Dis. 164, 686–692. Deyde, V.M., Khristova, M.L., Rollin, P.E., Ksiazek, T.G., Nichol, S.T., 2006. CrimeanCongo hemorrhagic fever virus genomics and global diversity. J. Virol. 80, 8834–8842. Elata, A.T., Karsany, M.S., Elageb, R.M., Hussain, M.A., Eltom, K.H., Elbashir, M.I., Aradaib, I.E., 2011. A nosocomial transmission of Crimean-Congo hemorrhagic fever to an attending physician in north Kordufan, Sudan. Virol. J. 8, 303 http:// www.virologyj.com/content/8/1/303 Ergonul, O., 2006. Crimean-Congo haemorrhagic fever. Lancet Infect. Dis. 6, 203–214.
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Original article
Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan Alaa M. Ibrahim, Ibrahim A. Adam, Badreldin T. Osman, Imadeldin E. Aradaib ∗ Molecular Biology Laboratory (MBL), Department of Clinical Medicine, Faculty of Veterinary Medicine, University Khartoum, Sudan
a r t i c l e
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Article history: Received 7 August 2014 Received in revised form 24 November 2014 Accepted 4 March 2015 Available online xxx Keywords: Epidemiology Cattle Crimean-Congo hemorrhagic fevers ELISA Sudan
a b s t r a c t Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by CCHF virus (CCHFV) of the genus Nairovirus in the family Bunyaviridae. CCHFV causes subclinical infection in domestic livestock and an often fatal hemorrhagic illness in humans, with approximately 30% mortality rates. In the present study, a cross-sectional serosurvey was conducted in a total of 282 randomly selected cattle from five localities in East Darfur State, Sudan. The exposure status to CCHF was determined using enzyme-linked immunosorbent assay (ELISA) for detection of CCHFV-specific IgG antibodies in cattle serum samples. The CCHFV-specific IgG antibodies were detected in 54 out of 282 animals, accounting for a 19.14% prevalence rate. Older cattle (>2 years of age) were approximately five times more likely to be infected with the virus (OR = 4.90, CI = 1.28–18.98, p-value = 0.02). Heavily tick-infested cattle (ticks all over the body) were at 11 times higher at risk compared to tick-free animals (OR = 11.11, CI = 2.86–43.25, p-value = 0.01). Grazing system is another factor affecting CCHF, where cattle grazing on open system were 27 times more at risk compared to other grazing systems (OR = 27.22, CI = 7.46–99.24, p-value = 0.001). There was an association between localities and CCHF cattle (OR = 0.24, CI = 0.07–0.83, p-value = 0.02). This study confirms the exposure of cattle to CCHF in East Darfur and identifies potential risk factors associated with the disease. Further epidemiological studies and improved surveillance are urgently needed to prevent a possible outbreak of CCHF among humans in the Darfur region of Sudan. © 2015 Elsevier GmbH. All rights reserved.
Introduction Crimean-Congo hemorrhagic fever (CCHF), caused by CCHF virus (CCHFV), is a zoonotic tick-borne disease, which is transmitted to humans by tick bites, handling of ticks, exposure to blood or tissues of viremic livestock, or direct contact with blood and bodily fluids of infected patients (Swanepoel et al., 1987; Vesenjak-Hirjan et al., 1991; Schwarz et al., 1995, 1996; Rodriguez et al., 1997). CCHF outbreaks constitute a threat to public health because of its epidemic potential, the severity of disease it causes, its potential to cause nosocomial outbreaks, and the limited options available for treatment and management of infected patients (Altaf et al., 1998; Burt et al., 1998; Ergonul, 2006; Whitehouse, 2004; Avsic-Zupanc, 2007). Recently, CCHFV has been repeatedly reported as an important emerging infectious viral pathogen in the Kordufan region, Sudan. In Sudan, the first outbreak of CCHF was reported in 2008 among health care workers in Alfulah rural hospital, West Kordufan (Aradaib et al., 2010). Subsequently, another outbreak was
∗ Corresponding author. Tel.: +249 912380932; fax: +249 185312638. E-mail address: [email protected] (I.E. Aradaib).
reported in 2009 in Donkup village, Abyei District, South Kordufan (Aradaib et al., 2011). In February, 2010, a nosocomially acquired CCHFV infection was reported in an attending physician in North Kordufan as a result of providing medical care to CCHF infected patient from Lagawa District, an area of endemicity in the State of South Kordufan. However, CCHF has never been recognized in the Darfur region of the Sudan (Elata et al., 2011). Serological studies have suggested the presence of various arboviruses in Sudan, including CCHFV (Watts et al., 1994; McCarthy et al., 1996). Indirect serological evidence of CCHF infection was recorded in camels exported from Sudan to Egypt (Morrill et al., 1990) and in sheep and goats exported to Saudi Arabia (Hassanein et al., 1997). Recently, we reported an overall prevalence rate of 7% among cattle in the State of North Kordufan (Adam et al., 2013). It is well documented that viremia and CCHFV-specific antibodies develop in infected livestock including, sheep, cattle and camels as well as in domestic donkeys (Swanepoel et al., 1987; Lwande et al., 2012). However, the infection is subclinical and no clinical hemorrhagic disease appears to be associated with CCHFV in infected animals (Burt et al., 1993). Nevertheless, cattle can amplify the virus and will become seroconverted. However, they do not show any signs of clinical hemorrhagic disease indicating
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subclinical infection. Therefore, at the time of viremia infected cattle could provide a potential source of infection to infested ticks. In addition, viremic cattle could also provide infection to humans, particularly after slaughtering and subsequent contact with their infected tissues including blood or meat. Therefore, it is suggested that these animals play an important role in the epidemiology of the disease (Shepherd et al., 1987; Garcia et al., 2006; Martin et al., 1987). Currently, it is becoming increasingly obvious that the control of emerging viral pathogens, such as CCHFV, is especially important in the Sudan given the large numbers of livestock in the country, and their importance to the national economy and rural communities. To better predict and respond to CCHF in Darfur region, further epidemiologic studies including implementation of improved surveillance, for such an emerging viral pathogen, are urgently needed (Martin et al., 1987). The present study was conducted to estimate the prevalence and to identify the potential risk factors associated with CCHF among cattle in East Darfur State, Sudan. Materials and methods Study area The State of East Darfur is located between latitude 12–9.5 N–longitude 25–28 E and altitude of 449 m (1476 feet) above sea level and about 831 km from Khartoum, the capital of Sudan. The State is boarded by North Darfur State in the north; Northern Kordufan in the east, South Darfur in the west, and is bordered by the State of South Sudan to the south. Total population in East Darfur State is approximately 0.3 million as estimated in 2006. The livestock population in East Darfur constitutes one of the major sources of the income to rural communities and the national economy, at large. East Darfur State is one of the main animal rearing areas in the country and possesses large numbers of live stock. The cattle population in this state is presently estimated to be as 2–3 million, 80% of which are kept under nomadic system, where nomads migrate continuously to south in the dry season and back to north in the rainy season. During this movement the nomads cover areas at the borders of the State of South Sudan up to north Darfur, where the animals inter mix freely and share common water sources, pasture route and premises with cattle in the State of South Sudan. The map of the State of East Darfur is shown in Fig. 1. Study design A cross sectional study was conducted to estimate the prevalence rate of CCHFV group-specific IgG antibodies in cattle and to investigate potential risk factors associated with the disease. The multistage probability sampling method was conducted. Five localities were randomly selected from the State of East Darfur. Two administration units were selected from each locality. Seven villages were selected from each unit. Finally, simple random sampling was applied to choose the animals from each herd. Questionnaire A pre-tested structured questionnaire with the primary objective of elucidating the multifactorial background of disease was conducted in an interactive manner at all selected herds. All animals included in this study were subjected to a questionnaire, which was filled out by the animal owners. The questionnaire included individual risk factor attributes including age (younger animals <2 years, older animals 2 years and above), sex (male, female), breed (indigenous, cross), body condition (thin, emaciated, fat), and management risk factor attributes including herd size (small, medium,
Fig. 1. Map of the State of East Darfur, Sudan.
large), grazing system (nomadic, semi-nomadic, stationary), tick vector (presence or absence), the source of each animal in the herd (raised on farm, purchased from other farms or purchased from local market) and the five localities included in the study. Ethical clearance The blood collection procedure from cattle was performed by qualified veterinarians following proper physical restraint of animals to ensure both personnel and animal safety. Livestock owners were explained the study purposes and procedures and upon agreeing to participate, they provided a written consent prior to study procedures and blood collection from their animals. The study received ethical clearance from the Research Board of the College of Veterinary Medicine, Sudan University for Science and Technology, Khartoum, Sudan. The risk factor information was obtained from the animal owners through the questionnaire form, which permitted use of the blood samples for diagnostic and research purposes. Collection of blood samples A total of 282 blood samples of cattle were collected randomly from five localities in East Darfur State, Sudan. These localities include (ElDdeain, AbuJabra, Assalaya, Elfirdus, and Bahr Alarab). Blood samples were collected by qualified veterinarians from the jugular veins in clean sterile vaccutainers and were allowed to clot and sera were separated and kept frozen at −20 ◦ C until used. Enzyme-linked immunosorbent assay (ELISA) Indirect enzyme-linked immunosorbent assay (ELISA) was performed to screen the sera for CCHFV-specific IgG antibodies basically as described (Adam et al., 2013). ELISA was performed in 96-well immunoassay microplates (Nunc, Roskilde, Denmark) and optimal working dilutions of reagents were determined by
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chessboard titration. Unless stated otherwise, 100 uL test volumes were used. Incubations were performed for 1 h at 37 ◦ C. The plates were washed three times with PBS containing 1.0% Tween 20 (Merck, Darmstadt, Germany) (PBST), wells were post-coated with 200 l of PBS containing 2% bovine serum albumin (Calbiochem, La Jolla, USA), and the diluents for the reagents was PBS containing 10% Skimmed milk (Amba, Denmark). Briefly, the plates were coated with sucrose-acetone extracted CCHFV antigen and incubated overnight at 4 ◦ C. The source of the antigen used is cell lysate from CCHFV Nigeria strain IbAr 10200. The antigen used in this study was obtained from the Center for Disease Control and Prevention, Atlanta, USA). The plates were washed, and aliquots of test sera (positive and negative controls) were added in separate wells at a dilution of 1:40. After a 1-h incubation, the plates were washed, and rabbit anti-bovine IgG conjugated with horse radish peroxidase (HRP) was added to the plate at a dilution of 1: 2000 and incubated for 1 h. The plates were then washed and the substrate, 2,22-azino-bis (3-ethylbenthiazoline-6-sulfonic acid (Kirkegaard and Perry Laboratories) was added. A positive CCHFV-infected cattle serum sample was incorporated in each ELISA plate as positive control to estimate the higher limit of the sensitivity. Negative control sera were obtained from CCHF-free animals and from cattle infected with Rift Valley fever virus (RVFV), a related viral hemorrhagic fever virus, to estimate the lower level of the specificity of the ELISA assay. The results were read using ELISA reader set at 405 nm. A presumptive diagnosis was made when IgG antibodies in the test sample had a significant color change or had higher optical density than the ratio between the positive and negative controls. Statistical analyses The data collected were entered into a computer on a Microsoft Excel spreadsheet. Statistical analysis was performed using ‘Statistical package for the social sciences’ (SPSS), version 16 (for Windows). Associations between the outcome variable (status of CCHFV-specific antibodies in cattle) and its potential risk factors were first screened in a univariable analysis using Chi-square test. Potential risk factors with p-value <0.25 (two tailed; ˛ = 0.25) were initially considered significant in 2 test. The results of the univariable analysis were reentered in the final model using multivariable analysis. A multivariable model for the outcome variable was constructed using manual stepwise forward logistic regression analysis. CCHF was considered as the dependent variable and the risk factors as independent variables. Finally, odd ratios and 95% confidence interval (CI) were calculated, and risk factors with a p-value <0.05 were taken as significant association to CCHFV infection. Results The result of this survey showed that 54 out of 282 cattle serum samples were found to be positive for CCHFV-specific-IgG antibodies, accounting for an overall prevalence rate of a 19.14% among cattle in East Darfur State, Sudan. Univariate analysis using Chi-square test was conducted for the association between the potential risk factors and CCHFV infection and p-value (p ≤ 0.25) was initially considered significant. The results of the univariate analysis showed that the independent variables including, age, animal source, tick number, body condition, grazing system, herd size, tick treatment frequency and locality were statistically significant (Table 1). The risk factors that were significant in the univariable model were re-entered into a final multivariate model using logistic regression analysis. In the final models, a variable with a p-value <0.05 was considered statistically significant. The individual risk factors attributes indicated that the older cattle (>2 years of age)
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were approximately five times more likely to be infected with the virus (OR = 4.90, CI = 1.28–18.98, p-value = 0.02). There was an association between locality (Barelarab) and CCHFV infected cattle (OR = 0.24, CI = 0.07–0.83, p-value = 0.02). The management risk factors attributes showed that heavily tick-infested cattle (ticks all over the body) were eleven time higher at risk compared to tick-free animals (OR = 11.11, CI = 2.86–43.25, p-value = 0.01). In addition, the type of the grazing system has significant association with CCHFV seropositivity. Cattle grazing on an open system were 27 times more likely to be at risk for CCHF compared to those grazing on a close system (OR = 27.22, CI = 7.46–99.24, p-value = 0.001). The results are summarized in (Table 2). In contrast, there was no significant association between CCHFV seropositive cattle and other individual or management risk factors included in the study such as animal sex, breed, body condition, animal source, herd size and tick treatment.
Discussion Crimean-Congo hemorrhagic fever is a tick-borne viral disease mainly affecting nomads who live in close contact with tickinfested livestock, namely Hyalomma ticks, the principal vectors of the virus. CCHFV causes an often fatal hemorrhagic illness in humans and asymptomatic infection in domestic livestock. In rural communities, tick-infested donkeys were considered to be potential source of CCHF in humans (Lwande et al., 2012). Therefore, it is suggested that livestock and domestic donkeys play an important role in the epidemiology of the disease. The occurrence of the periodic outbreaks and sporadic cases of CCHF in endemic countries necessitate the importance of improved surveillance system for this important emerging viral pathogen (Altaf et al., 1998; Aradaib et al., 2010, 2011; Burney et al., 1980; Ahmeti and Raka, 2006; Gurbuz et al., 2009; Khan et al., 1997). In this study, cattle were selected as CCHFV infection appears to occur most frequently in larger mammals, which are the preferred hosts of adult tick vector, Hylomma species (Shepherd et al., 1998). We believe the occurrence of multiple CCHF outbreaks in region in the past few years, and the risks these cases pose for medical staff in resource poor health care facilities indicate the importance of improved surveillance in Sudan for this important disease. The Darfur region of Sudan was thought to be CCHF-free zone and infection with CCHFV has never been reported in the region. This study expands on existing data indicating that CCHF, known to be endemic in Africa, is broadly distributed within Sudan. In addition, our investigation illustrates that CCHF has been reported for the first time among cattle in the Darfur region, Sudan. More over, collection of bovine blood samples for generation of the present data was made available despite the violence in the region. It is worth mentioning that Darfur region shares boarders with four different countries including Chad, Libya, Central African Republic and State of South Sudan and it would be interesting to trace the movement of the virus in the region. Further more, the results of this epidemiological study will stimulate further molecular characterization studies such as viral genome sequencing and subsequent phylogeny to determine the virus genetic lineages circulating in the region. Our result indicated that, the overall prevalence of CCHFV IgG-specific antibodies was 19.14%. The CCHFV IgG-specific antibodies recorded in this study showed evidence of prior exposure to CCHF by cattle population in East Darfur State, Sudan. The high prevalence rate (19.14%) indicates significant circulation of CCHFV among cattle of East Darfur and that human population in the region may have also been infected with CCHFV. However, acute CCHF cases have never been diagnosed among humans in Darfur region of the Sudan. This is probably attributed to the fact that
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Table 1 Univariate analysis for the association between potential risk factors and CCHFV infection among cattle in East Darfur State of Sudan using Chi-square test. Risk factors
Animals tested
Locality Aldeain Abu Jabra Bahr Alarab Assalaya Elfirdus
Animals affected (%)
74 49 99 23 37
16 (21.6%) 9 (18.4%) 13 (13.1%) 4 (17.4%) 12 (32.4%)
Age Small Old
67 215
3 (4.5%) 51 (23.7%)
Sex female male
220 62
42 (19.1%) 12 (19.4%)
Breed Endigenous Cross
265 17
52 (19.6%) 2 (11.8%)
Body condition Fat Thin
20 262
2 (10%) 52 (19.8%)
Animal source -Raised on farm Purchased from other farm Purchased from local market
206 50 26
35 (17%) 12 (24%) 7 (26.9%)
Grazing system Stationary Nomadic
112 170
3 (2.7%) 51 (30%)
Herd size Small Medium Large
48 119 115
5 (10.4%) 25 (21%) 24 (20.4%)
Tick treatment frequency 1–3 months More than 3 months
80 202
18 (22.5%) 36 (17.8%)
Tick number No Small Large
75 112 85
5 (6.7%) 24 (19.7%) 25 (29.4%)
Table 2 Multivariate analysis using logistic regression model for significant association (p < 0.05) between risk factors and CCHFV infection among cattle in East Darfur State, Sudan. Risk factors
OR
95% CI
p-value
Age
Small Old
Ref 4.90
1.28–18.98
0.02
Localities
Aldeain Abugabra Bahrelarab Asalaya Fardus
Ref 0.65 0.24 0.75 1.51
0.18–2.44 0.07–0.83 015–3.89 0.42–5.50
0.52 0.02 0.73 0.53
Stationary Nomadic
Ref 27.22
7.46–99.24
0.01
No Small Large
Ref 6.05 11.11
1.72–21.33 2.86–43.25
0.01 0.01
Grazing system Tick number
several acute febrile illnesses such as malaria, brucellosis, typhoid and other related hemorrhagic fevers are endemic in the region. Under these circumstances, it would be extremely difficult, if not impossible, for health care workers to differentiate CCHF from other endemic febrile illness based on clinical presentation. Recently, a lot of research efforts have been conducted in our laboratory to facilitate rapid detection and differentiation of arbovirus infections with special emphasis on CCHF during outbreaks of the disease in the Kordufan region of Sudan (Aradaib et al., 1998, 2005, 2010, 2011,
df
2
p-value
4
6.89
0.14
1
12.23
0.01
1
0.01
0.55
1
0.64
0.33
1
1.16
0.22
2
2.40
0.13
1
32.55
0.01
2
2.85
0.24
1
0.81
0.23
2
13.35
0.01
2013). Previous CCHF studies in humans showed prevalence rate of 13.1% among nomadic tribes in Senegal (Chapman et al., 1991). In addition, a serosurvey on CCHF among pastoralists in Ijara District, east Northern province, Kenya, showed seroprevalence rates of 23 and 14% in Sangailu and Ijara regions, respectively (Lwande et al., 2012). Using the multivariate logistic regression analysis, only four independent risk factors were associated with CCHFV seropositivity in cattle and were considered statistically significant p-value <0.05. There was significant difference between CCHFV infection rate and the age of the animal. When assessing age as risk factor, it was shown that the calves started to get infected with CCHFV after the age of 2 years. At this age, the animals are usually released into the pasture for grazing, where they are likely to be exposed to infected tick and subsequent CCHFV infection. We believe that the association of CCHFV infection rate and age is probably attributed to frequent exposure of older cattle to infected tick in the pasture. In contrast, young calves are usually kept indoors and are well taken care of by the animal owners from infectious diseases particularly, tick-borne infections. It should be noted that the CCHFV-specific antibodies detected among cattle in East Darfur state indicate natural infection as there is no vaccination program for this disease. In addition, all cattle included in this study aged over one year. Therefore, it is assumed that maternal antibodies no longer persisted and that antibody indicated direct exposure to CCHFV. Previous epidemiological surveys indicated that there were higher risks of older cattle for CCHF in different countries including Sudan, Egypt, Iran and Turkey (Adam et al., 2013; Garcia et al., 2006; Mohamed
Please cite this article in press as: Ibrahim, A.M., et al., Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.002
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et al., 2008; Lotfollahzadeh et al., 2011). In addition, the study revealed significant association between heavily tick-infested cattle and CCHFV seropositivity. It is well documented that CCHF is a tick-borne zoonotic disease and that heavily tick-infested cattle are likely to become CCHFV positive by bites of infected ticks (Adam et al., 2013; Mohamed et al., 2008; Swanepoel et al., 1985). Moreover, there was significant association between the open system of grazing and CCHFV seropositive cattle. Frequent exposure of cattle to the pasture results in contact with many tick-infested species of mammals, which can transmit the virus (Swanepoel et al., 1983). It should also be noted that treatment of cattle with insecticides should be applied monthly to prevent tick infestation. Small vertebrates such as hares and hedgehogs, which are infested by immature ticks, may be particularly important as amplifying hosts (Swanepoel et al., 1987). The ground-based birds such as ostrich can amplify the virus; others are refractory to infection but may act as mechanical vectors by transporting infected ticks over a long distance. Migratory birds might spread the virus between distant geographic areas (Deyde et al., 2006; Lindeborg et al., 2011). Furthermore, there was a significant difference between localities and CCHFV seropositivity. The highest and lowest rates of CCHFV seropositivity were recorded in Bahr Elarab and Asalaya, respectively. The high level of CCHFV infection in Bahr Elarab is attributed to the dry climate and high variations in temperatures in the region. In contrast, the risk assessment studies indicated that there was no significant association between CCHF and the rest of the individual or management risk factors included in the study. It is worth mentioning that the gender has no significant association with CCHFV seropositivity as both sexes are equally infected with the disease. The lack of association between CCHF and tick treatment is probably due to the infrequent treatments with insecticides by the nomads specially, under field conditions. Conclusions This study confirms the transmission of CCHFV among cattle in East Darfur State. Age, locality, open system of grazing, and tick infestation are potential risk factors for CCHF. Human populations in East Darfur State are at risk of contracting the infection with the virus. The residents should also be educated about the risks of the disease, and prevention of the infection through tick control. In this regard, effective animal husbandry and management system should be applied to control tick infestations in domestic livestock populations. Health care providers should be aware of the disease and be prepared for a possible outbreak in the region. Physicians and medical health workers in East Darfur State should consider this virus in their efforts to diagnose the disease in patients with clinical presentations compatible with those of CCHF. Future surveillance program for CCHF should be extended to include other susceptible animals such as sheep, goats, camels and donkeys. In addition, the high-risk human groups (Veterinarians, animal attendants, slaughter house workers and butchers), and the distribution of ticks in the region should also be considered to better predict and respond to CCHF outbreak in the State of East Darfur, Sudan. Author details Molecular Biology Laboratory (MBL), Department of Clinical Medicine, Faculty of Veterinary Medicine, University of Khartoum, P.O. Box 32 Khartoum North, Sudan. Authors’ contributions AMI designed the study, optimized the ELISA for detection of IgG antibodies in cattle sera, performed the statistical analysis, and
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prepared the draft manuscript. IAA collected blood samples and designed the study. IEA designed the experiment and prepared the final manuscript. All authors read and approved the final version of the manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgements The authors would like to thank Dr. Stuart T. Nichol of the Molecular Biology Laboratory, Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, USA, for provision of CCHFV antigen. The technical assistance of Dr. Shakir Z. Bushara; Dr. Mubarak Mahmoud and Mr. Abdalla M. Fadlelmoula is gratefully acknowledged. This study received partial financial support from the University of Khartoum, Sudan. The result of this study does not reflect the opinion of the funding sources. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.03.002 References Adam, I.A., Mahmoud, M.A.A., Aradaib, I.E., 2013. A seroepidemiological survey of Crimean Congo hemorrhagic fever among Cattle in North Kordufan State, Sudan. Virol. J. (10), 178, http://dx.doi.org/10.1186/1743-422X-10-178 Ahmeti, S., Raka, L., 2006. Crimean-Congo haemorrhagic fever in Kosova: a fatal case report. Virol. J. 3, 85. Altaf, A., Luby, S., Ahmed, A.J., Zaidi, N., Khan, A.J., Mirza, S., Mc Cormick, J., FisherHoch, S., 1998. Outbreak of Crimean-Congo haemorrhagic fever in Quetta, Pakistan: contact tracing and risk assessment. Trop. Med. Int. Health 3, 878–882. Aradaib, I.E., Erickson, B.R., Karsany, M.E., Khristova, M.L., Elageb, R.M., Mohamed, M.E.H., Nichol, S.T., 2011. Multiple Crimean-Congo hemorrhagic fever virus strains are associated with disease outbreaks in Sudan, 2008–2009. PLoS Negl. Trop. Dis. 5, e1159, http://dx.doi.org/10.1371/journal.pntd.0001159 Aradaib, I.E., Erickson, B.R., Mustafa, M.E., Khristova, M.L., Saeed, N.S., Elageb, R.M., Nichol, S.T., 2010. Nosocomial outbreak of Crimean-Congo hemorrhagic fever, Sudan. Emerg. Infect. Dis. 16, 837–839. Aradaib, I.E., Mohamed, M.E.H., Abdalla, T.M., Abdalla, M.Y., Abdalla, M.A., Sarr, J.A., Karrar, A.E., 2005. Serogrouping of United states and some African serotypes of Bluetongue virus using RT-PCR. Vet. Microbiol. 111, 125–128. Aradaib, I.E., Mohammed, M.E.H., Ahmed, S., Ibrahim, K.E.E., Yilma, T.D., Karrar, A.E., Cullor, J.S., Osburn, B.I., 1998. A multiplex PCR for simultaneous detection and identification of United States serotypes of Epizootic haemorrhagic disease virus. Sud. J. Vet. Sci. Anim. Husb. 37, 1–12. Aradaib, I.E., Erickson, B.R., Karsany, M.S., Khristova, M.L., Elageb, R.M., Mohamed, M.E., Nichol, S.T., 2013. Rift Valley fever, Sudan, 2007–2010. Emerg. Infect. Dis. 19, 45–52. Avsic-Zupanc, T., 2007. Epidemiology of Crimean-Congo hemorrhagic fever in the Balkans. In: Ergonul, O.W.C.A. (Ed.), Crimean-Congo Hemorrhagic Fever: A Global Perspective. Springer, Dordrecht, pp. 75–88. Burney, M.I., Ghafoor, A., Saleen, M., Webb, P.A., Casals, J., 1980. Nosocomial outbreak of viral hemorrhagic fever caused by Crimean hemorrhagic fever-Congo virus in Pakistan in 1976. Am. J. Trop. Med. Hyg. 29, 941–947. Burt, F.J., Leman, P.A., Smith, J.F., Swanepoel, R., 1998. The use of a reverse transcription-polymerase chain reaction for the detection of viral nucleic acid in the diagnosis of Crimean-Congo haemorrhagic fever. J. Virol. Methods 70, 129–137. Burt, F.J., Swanepool, R., Braack, L.E.G., 1993. Enzyme-linked immunosorbent assays for the detection of antibody to Crimean-Congo haemorrhagic fever virus in the sera of livestock and wild vertebrates. Epidemiol. Infect. 111, 547–557. Chapman, L.E., Wilson, M.L., Hall, D.B., 1991. Risk factors for Crimean-Congo hemorrhagic fever in rural northern Senegal. J. Infect. Dis. 164, 686–692. Deyde, V.M., Khristova, M.L., Rollin, P.E., Ksiazek, T.G., Nichol, S.T., 2006. CrimeanCongo hemorrhagic fever virus genomics and global diversity. J. Virol. 80, 8834–8842. Elata, A.T., Karsany, M.S., Elageb, R.M., Hussain, M.A., Eltom, K.H., Elbashir, M.I., Aradaib, I.E., 2011. A nosocomial transmission of Crimean-Congo hemorrhagic fever to an attending physician in north Kordufan, Sudan. Virol. J. 8, 303 http:// www.virologyj.com/content/8/1/303 Ergonul, O., 2006. Crimean-Congo haemorrhagic fever. Lancet Infect. Dis. 6, 203–214.
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Please cite this article in press as: Ibrahim, A.M., et al., Epidemiological survey of Crimean Congo hemorrhagic fever virus in cattle in East Darfur State, Sudan. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.002